The goal of this project is to understand the role of histone macroH2A (mH2A) in X chromosome inactivation. mH2A is an unusual core histone that contains an H2A domain and a large nonhistone domain. It is present in many mammalian cells and replaces conventional H2A in a subset of nucleosomes - typically less than 5 percent of the nucleosomes contain mH2A. mH2A is preferentially associated with the inactive X chromosome (Xi) of female mammals. This suggests that it has a role in silencing transcription through chromatin structure. Several approaches will be used to examine mH2A function. Immunofluorescence with mH2A antibodies and fluorescence in situ hybridization (FISH) will be used to examine the timing of mH2A accumulation in Xi. These studies will help determine whether mH2A is involved in the initiation of X- inactivation, or maintenance of the inactive state after it is established. To directly examine the role of mH2A in X-inactivation we will examine the phenotypic effects of mutating a mouse mH2A gene. This will be accomplished by using homologous recombination to mutate the mH2A1 gene of mouse embryonic stem cells. Mutated cells will be injected into blastocyst stage mouse embryos to produce mice with a mutated mH2A gene. Breeding of these mice will allow us to assess the effects of the absence of mH2A1 proteins on X-inactivation and mouse development. Human mH2A genes will be mapped to specific chromosomal loci to assess their possible involvement in human genetic diseases. The functional properties of the nonhistone domain will be examined to assess the role of this region in mH2A function. The ability of this domain to interact with DNA, RNA, and nuclear proteins will be examined using direct binding assays and by interactive cloning methods such as yeast two-hybrid and phage display. Understanding the function of mH2A might give insights into human disorders such as cancer that involve disruptions of gene regulation. Better understanding of chromatin mediated gene repression could also be useful for gene therapy, where potentially therapeutic genes are sometimes inactivated after introduction into target cells.